Near-infrared noninvasive spectroscopic determination of pH

ABSTRACT

Methods and apparatus for, preferably, determining noninvasively and in vitro pH in a human. The non-invasive method includes the steps of: generating light at three or more different wavelengths in the range of 1000 nm to 2500 nm; irradiating blood containing tissue; measuring the intensities of the wavelengths emerging from the blood containing tissue to obtain a set of at least three spectral intensities v. wavelengths; and determining the unknown values of pH. The determination of pH is made by using measured intensities at wavelengths that exhibit change in absorbance due to histidine titration. Histidine absorbance changes are due to titration by hydrogen ions. The determination of the unknown pH values is performed by at least one multivariate algorithm using two or more variables and at least one calibration model. The determined pH values are within the physiological ranges observed in blood containing tissue. The apparatus includes a tissue positioning device, a source, at least one detector, electronics, a microprocessor, memory, and apparatus for indicating the determined values.

This invention is made with U.S. Government support and the U.S.Government has certain rights in this invention.

This application is a continuation-in-part of application Ser. No.08/257,875 filed on Aug. 12, 1994, now U.S. Pat. No. 5,630,413, which isa continuation of 07/910,004 filed on Jul. 6, 1992, now U.S. Pat. No.5,355,880.

BACKGROUND OF THE INVENTION

This invention relates to both methodology and apparatus for thenoninvasive determination of hydrogen ion concentration ( H⁺ !),commonly reported as -log ( H⁺ !) or pH in tissue.

Current methodology for blood pH measurement requires blood samples tobe measured using a sophisticated blood analyzer equipped withelectrodes to measure the desired analyte. Standard analyzerinstrumentation (such as supplied by Ciba Corning, Abbot Laboratoriesand Radiometer) automates sample preparation, delivery, and measurement.After each sample is analyzed, the electrodes must be thoroughly washedto prevent protein buildup on the electrode surfaces, and the analyzermust be calibrated at a minimum of every two hours. Measurement of pH,along with other blood analytes (i.e., PCO₂, HCO⁻ ₃ !, PO₂ and O₂ sat.),can be made with these analyzers in approximately two minutes. However,due to the size and expense of commercially available blood analyzers,such equipment is kept in central locations in most hospitals, requiringthe blood sample to be transported to the analyzer.

While an individual analysis can be made in a few minutes, for anindividual patient (often critically ill) the process is far fromcontinuous and, because it is invasive, not without discomfort or pain.First, arterial blood has to be withdrawn. Immediately after withdrawal,the sample is placed on ice to inhibit red blood cell metabolism, whichmetabolism would alter the sample's blood gas parameters and lead to anincorrect measurement of the patient's blood gas values (including pH).The sample is then, typically, transported to the clinical chemistrylaboratory in the hospital, where it is logged in. Next, the sample isthen analyzed by conventional electrochemical techniques with the typeof equipment identified above. Finally, the results are entered in thehospital computer and made available to the physician forinterpretation. Thus, the analysis can require a significant period oftime (approximately 30 minutes) during which patient status can change.

Localizing pH measurement to treatment rooms, with the use ofreagentless measurement of pH, would allow for semi-continuousmeasurement. This decrease in turnaround time for pH measurement would,in turn, provide the clinician better evaluation of treatment methods.While such improvements in pH measurement could be realized within-vitro optical methodologies, greater gains can be realized with thenoninvasive, in-vivo methodology as disclosed herein.

Several groups have published papers on the spectroscopic effects of pHvariation on blood, primarily related to the Bohr effect. See, S. K.Soni and L. A. Kiesow, "pH-Dependent Soret Difference Spectra of theDeoxy and Carbonmonoxy Forms of Human Hemoglobin and Its Derivatives,"Biochemistry, 16, 1165-1170, 1977, wherein the authors reported seeingvariation in the Soret absorption bands of hemoglobin (i.e., 350-400 nm)with pH. These spectral variations were ascribed to structural changesin the porphyrin of hemoglobin, caused by pH variation. Otherresearchers have found that the visible absorption bands (i.e., 400-550nm) from oxyhemoglobin have a pH sensitivity. See P. D. Wimberley, N.Fogh-Andersen, O. Siggaard-Andersen, F. C. Lundsgaard and W. G.Zijlstra, "Effect of pH on the Absorption Spectrum of HumanOxyhemoglobin: a Potential Source of Error in Measuring the OxygenSaturation of hemoglobin," Clinical Chemistry, 34, 750-754, 1988.Wimberley and coworkers did not attribute these spectral changes to theknown variation in oxygen affinity caused by pH variation (i.e., Bohreffect). Rather, the variations seen were attributed to changes in thesurrounding globin structure of the hemoglobin molecules, giving rise tochanges in the charge-transfer bands of the porphyrin.

In the mid-infrared region (i.e., from approximately 2500 nm-10,000 nm),work has been done to determine the effects of pH variation on thevibrational modes of hemoglobin. In-vitro experiments with purehemoglobin have shown that certain vibrational modes of the globins, inparticular, the sulfur-hydrogen (S-H) stretching vibration of twocysteinyl (αCys104, βCys112) are strongly hydrogen bonded and aresensitive to slight conformational changes of oxyhemoglobin caused by pHvariation. See S. E. Antri, O. Sire and B. Alpert, "Relationship BetweenProtein/Solvent Proton Exchange and Progressive Conformation andFluctuation Changes in hemoglobin," Euro. J. Biochem., 191, 163-168,1990. These S-H vibrational modes were used to study the quaternary andtertiary structure of the hemoglobin molecule. However, from a clinicalstandpoint the mid-IR region is not useful because of the very highabsorption of these wavelengths by tissue (i.e., there is no returnsignal from the tissue).

The near infrared region (i.e., from approximately 500 to 2500 nm) hasbeen an active area of research for those pursuing non-invasivedetection of blood analytes. Significant research has been done in theareas of oxygen saturation and glucose monitoring. See: (1) D. M.Haaland, M. R. Robinson, G. W. Koepp, E. V. Thomas and R. P. Eaton,"Reagentless Near-infrared Determination of Glucose in Whole Blood UsingMultivariate Calibration," Appl. Spec., 46, 1575-1578, 1992; (2) B.Chance, J. S. Leigh, H. Miyake, D. S. Smith, S. Nioka, R. Greenfeld, M.Finander, K. Kaufman, W. Levy, M. Young, P. Cohen, H. Yoshioka and R.Boretsky, "Comparison of Time-Resolved and -Unresolved Measurements ofDeoxyhemoglobin in Brain," Proc. Natl. Acad. Sci., 85, 4971-4975, 1988;(3) M. A. Arnold and G. W. Small, "Determination of Physiological Levelsof Glucose in an Aqueous Matrix with Digitally Filtered FourierTransform Near-Infrared Spectra," Anal. Chem., 62, 1457-1464, 1990; (4)Y. Mendelson and M. V. Solamita, "The Feasibility of SpectrophotometricMeasurements of Arterial Oxygen Saturation from the Fetal ScalpUtilizing Noninvasive Skin-Reflectance Pulse Oximetry," BiomedicalInstrumentation & Technology, May/June, 215-224, 1992; (5) U.S. Pat. No.4,975,581 to Robinson, et al.; and (6) U.S. Pat. No. 5,492,032 toRobinson, et al. The lowered extinction coefficients (e.g., the size orintensities of the bands) and decreased scatter coefficients in thenear-infrared region affords a much greater penetration depth (i.e.,1-10 mm) into human tissue than the visible or mid-infrared regions. Inthe near-infrared, absorbances are primarily due to overtone andcombination bands of fundamental stretching and bending vibrations,although some low lying electronic transitions appear at the shorterwavelengths. The strongest near infrared bands will arise when there isa strong asymmetry in the fundamental mode, such as exists with N--H,O--H and C--H vibrational modes, thus making these species the primaryabsorbers in the near infrared.

The most significant prior art on spectrograph measurement of pH is U.S.Pat. No. 5,355,880 to Edward V. Thomas, Mark R. Robinson, David M.Haaland and Mary K. Alam titled "Reliable Noninvasive Measurement ofBlood Gases" (hereinafter "Thomas et al.") discloses methods andapparatus for determining noninvasively and in vivo at least two of thefive blood gas parameters (i.e., pH, PCO₂, HCO₃ ⁻ !, PO₂ and O₂ sat.) ina human. The noninvasive methodology disclosed includes the steps of:generating light at three or more different wavelengths in the range of500 nm to 2500 nm; irradiating blood containing tissue; measuring theintensities of the wavelengths emerging from the blood containing tissueto obtain a set of at least three spectral intensities v. wavelengths;and determining the unknown values of at least two of pH, HCO₃ ⁻ !, PCO₂and a measure of oxygen concentration. The determined values were foundto be within the physiological ranges observed in blood containingtissues. The methodology disclosed also includes the steps of providingcalibration samples, determining if the spectral intensities v.wavelengths from the tissue represents an outlier, and determining ifany of the calibration samples represents an outlier. The determinationof the unknown values was performed by at least one multivariatealgorithm (e.g., PLS (partial least squares), PCR (principal componentregression) and CLS (classic least squares) using two or more variablesand at least one calibration model. Preferably, there is a separatecalibration for each blood gas parameter being determined. Themethodology can be utilized in a pulse mode and can also be usedinvasively. The apparatus disclosed by Thomas et al. includes a tissuepositioning device, a source, at least one detector, electronics, amicroprocessor, memory, and apparatus for indicating the determinedvalues.

The rationale of Thomas et al. for using multivariate analysis fornoninvasive blood gas determination is to enable accurate determinationof blood gas parameters where the information content in the spectraldomain utilized overlaps and where the infrared patterns for pH, PCO₂,PO₂ and HCO₃ ⁻ ! are small or do not exist in the absence ofinteractions with water and other blood or tissue components. This isespecially true for pH which does not exhibit a strong correlation inany specific region. Thomas et al. also observed that not only doeswater have exceptionally strong absorption bands in the near infraredregion, but H⁺ and O₂ have no absorption bands of their own in the nearinfrared.

Thomas, et al., disclose the measurement of arterial blood gases on alamb through the use of a Si array detector and a grating spectrometer.The spectra acquired were over the 500-1000 nm range. Thomas, et al., atcol. 28, ll. 28-35, went on to state:

Although the data used to demonstrate proof of concept in the lamb studywas recorded from 500 to 1000 nm, this is not the only frequency regionof interest. Specifically, the region from 1000 nm to 2400 nm containsinformation on both hydrogen ion concentration and CO₂, (E. Watson andE. H. Baughman, On-line analysis of caustic streams by near-infraredspectroscopy. Spectroscopy, Vol. 2, No. 1, pp. 44-48.

In the reference by E. Watson, et al., the spectroscopic variations dueto the varying hydrogen ion levels are due to changes in the waterabsorption bands. In summary, Thomas, et al., observed that hydrogenion, being an ion rather than a molecule, does not have infrared bands.However, hydrogen ions will bind to other species in solution that areinfrared active, thus a correlation for pH can be based on secondaryspectroscopic effects. In the wavelength region of 1000-2500 nm, Thomas,et al., do not specify the exact source of the spectroscopic informationfor pH measurements in blood and no figures were provided disclosing thespectroscopic bands of importance.

In this application, the dominant source of spectroscopic informationfor pH measurement in blood in the 1000-2500 nm is identified, figuresare provided demonstrating the relevant spectroscopic features, and thesource of spectroscopic information is found to be different thanidentified by E. Watson.

It is an object of the invention to determine pH both in blood in vitroand in vivo in human tissue, utilizing spectral data from histidine inthe range of 1000-2500 nm.

It is an object of the present invention to determine pH, both in bloodin vitro and in vivo in human tissue, utilizing the spectral data fromhistidine in one or more of the ranges: 1000-1300 nm; 1500-1820 nm;2040-2380 nm; and 1300-2380 nm.

It is a further object of the present invention to determine pH, both inblood and in vitro and in vivo in human tissue, utilizing spectral datafrom histidine at, approximately, one or more of the followingwavelengths: 1042 nm; 1111 nm; 1163 nm; 1600 nm; 2060-2115 nm; 2160 nm;2225-2235 nm; and 2360 nm.

A further object of the present invention is to provide a methodology,and associated apparatus, for determining pH, in blood in vitro and invivo in human tissue, with standard errors or prediction below 0.05 pHunits for a pH range of 1 (i.e., 6.8-7.8).

SUMMARY OF THE INVENTION

This invention relates to methodology and apparatus for determiningnoninvasively and in vivo pH in humans utilizing tissue spectra whichspectra contains histidine information. The method includes the steps ofgenerating light at three or more different wavelengths, the wavelengthsbeing in the range of 1000 nm to 2500 nm; noninvasively irradiatingblood containing tissue with the wavelengths so that there isdifferential attenuation of at least some intensities of thewavelengths, the wavelengths dependent differential attenuation being anfunction of the blood containing tissue, including histidine in theblood containing tissue; measuring the intensities of the wavelengthsemerging from the blood containing tissue to obtain a set of at leastthree spectral intensities v. wavelengths; estimating the value of thepH from the intensities emerging from the blood containing tissue byutilizing wavelength dependent differential attenuation derived from thehistidine, the value being within the physiological ranges observed inblood containing tissue. The method utilizes wavelength dependentdifferential attenuation derived from histidine which exhibits asensitivity due to changes in pH. The wavelengths used for theestimation of pH are those known to exhibit a differential attenuationdue to hydrogen ions reacting with histidine. The wavelengths are in therange of 1000-2500 nm. In the 1000-2500 nm region, wavelengths includeone or more of 1042, 1111 and 1163 nm. In the range of 1500-1820 nm, thewavelengths include 1600 nm. Finally, in the range of 2040-2500 nm thewavelengths include one or more of 2060-2115, 2160, 2225-2235 and 2360nm.

A quantitative analysis instrument for noninvasive spectroscopicmeasurement of pH in human tissue includes: a source of at least threedifferent wavelengths of light, the wavelengths being in the range of1000-2500 nm, at least some of the wavelengths having a wavelengthdependent differential attenuation due to histidine; optics fordirecting the wavelengths of light into the blood containing tissue; atleast one detector for measuring the intensity of at least a portion ofthose of the wavelengths of light emerging from the blood containingtissue that are differentially attenuated by histidine; electronics forprocessing the measured intensities to estimate pH values in tissue; andapparatus for indicating the estimated values of blood pH. Theelectronics utilizes a multivariate algorithm using at least twovariables and a calibration model. The algorithm is capable of usingmore spectral variables per sample than the number of calibrationsamples used to generate the model, and wherein each of the variablesconsists of at least one measured spectral intensity.

The invention also relates to methodology and apparatus for determiningin vitro pH in blood utilizing spectra which spectra contains histidineinformation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the relationship between pH and HCO₃ ⁻ ! is minimizedwith PCO₂ varying between 5 and 50 mmHg;

FIG. 2 illustrates how the correlation between pH and O₂ sat isminimized by varying PO₂ ;

FIG. 3 is a diagram of the flow system used for the spectral analysis oflysed blood and blood plasma;

FIG. 4 is a graph of absorbance v. wavelength (nm) for thirty-ninespectra of lysed blood solutions at varying pH, HCO₃ ⁻ ! and O₂ sat.,wherein the primary absorbances seen are those from water combinationbands;

FIG. 5 is a graph of the mean-centered spectral data for the thirty-ninelysed blood solutions, whose spectra are illustrated in FIG. 4;

FIG. 6 is a graph representing the correlation between reference pH(determined by conventional wet chemistry) v. predicted pH values usingPLS modeling in the 1500-1820 nm range;

FIG. 7 is a graph representing the correlation between reference pH(determined by conventional wet chemistry) v. predicted pH values usingPCR modeling in the 1500-1820 nm range;

FIG. 8 is a graph illustrating the relationship between the squaredcorrelation coefficient calculated between reference pH values andprinciple component analysis (PCA) scores, with the absorbance datacollected from titrated lysed blood in the 1500-1820 nm range;

FIG. 9 is a graph illustrating loading vectors v. wavelength (nm) forloading vector (5 for lysed blood) whose score best correlated to pHfrom spectral data collected from lysed blood, histidine, and plasmasolutions;

FIG. 10 is a graph representing the correlation between pH (determinedby conventional wet chemistry) v. predicted pH values using PLS Modelingin the 1000-1300 nm region;

FIG. 11 is a graph illustrating loading vectors v. wavelength (nm) forthe loading vector whose score best correlated to pH from spectral datafor whole blood and histidine solutions; and

FIG. 12 is a schematic illustration of the preferred embodiment of theapparatus of the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENT

As discussed above, spectroscopic detection of pH variation is dependenton a sensitive and appropriate indicator species. In blood, there arefour main systems responsible for buffering (i.e., combining withhydrogen or hydroxyl ions while maintaining a constant pH): hemoglobin;bicarbonate; plasma proteins; and phosphate. Each of these wasconsidered as the source of pH signal during the examination of lysedblood discussed below. Phosphate buffers are minimal in their bufferingcapacity, are in low concentration, provide essential no signal in thenear-infrared and, thus, were eliminated as a possible spectral sourceof pH information. Plasma proteins, while in significant concentration,do not provide a large variation in spectral signal to be a sufficientindicator species for pH. Support for this comes from the near-infraredspectra examined, which indicated only minimal correlation with pH.Titration data supports this finding. See, C. Tanford, S. A. Swanson andW. S. Shore, "Hydrogen Ion Equilibria of Bovin Serum Albumin," J. Amer.Chem. Soc., 77, 6414-6420, 1955.

The bicarbonate buffer is not an appropriate indicator species forspectroscopic determination of pH. The relationship between bicarbonateion and pH is defined by the chemical equilibrium:

    CO.sub.2 +H.sub.2 O⃡H.sub.2 CO.sub.3 ⃡HCO.sub.3.sup.- +H.sup.+

Thus, at constant CO₂, bicarbonate ion concentration is correlated pHthrough a simple logarithmic function and any spectroscopic variationdue to bicarbonate would be modeled as pH variation. Any variation inCO₂ content would break the correlation between bicarbonate and pH,causing a pH model based on bicarbonate concentration to predicterroneously. Perhaps more important is the clinical distinction betweenpH and bicarbonate measurements. Delineation of differing types ofalkalosis and acidosis are decided based on the relationship between pH,HCO₃ and CO₂ values. Thus, spectroscopic models for pH must not deriveinformation from the spectroscopic variation in bicarbonateconcentration. As discussed below, this is achieved by choosing thecalibration points in order to optimize orthogonality between pH andHCO₃ ⁻ !. In this manner, it is certain that if the bicarbonate ion hasa spectroscopic signature, it will vary orthogonally to the signaturefor pH and will not adversely affect the final pH model.

The hemoglobin buffer system is the largest component of the blood'sbuffering capacity, making it appropriate to use as an indicatorspecies. Hemoglobin derives its buffering capacity from histidinethroughout the molecule. It is known that variation in pH will cause achange in the tertiary structure of hemoglobin causing a correspondingchange in oxygen binding. Several amino acids which reside at the globinchain interfaces are thought to be largely responsible for thisstructure change, including histidine. Because of this, we havedetermined that, for robust spectroscopic model development, it isnecessary to break the direct correlation of hemoglobin oxygensaturation to pH. To achieve this, we titrated the so-called "Bohrprotons", causing the subsequent structure changes in the hemoglobinmolecule. However, we avoided making the spectroscopic model dependenton hemoglobin oxygen saturation through the use of the statisticaldesign set forth below.

To demonstrate that pH in human tissue can be accurately determinedutilizing spectral data from histidine in the range of 1300-2500 nm,fresh human blood, 500 ml, was collected from a healthy, nonsmokingvolunteer. An anticoagulant heparin (≅0.2 g/ml) was then added. The redblood cell walls were ruptured by sonicating 40 ml aliquots (i.e.,portions) of the blood using a sonication probe. The blood was kept inice water during the sonication process. The resulting solutions werecentrifuged at 3000 rpm and 10° C. Supernatant from each of thesolutions was removed, combined and used for sample preparation. Cellwalls were discarded.

To avoid the development of spectroscopic models for pH based onspurious and unreliable correlations, it is necessary to insureorthogonality between pH, HCO₃ ⁻ ! and O₂ sat. This was achieved using aLatin Hypercube with D-optimality criterion (i.e., a statistical designthat minimizes the possibility of results based on spuriouscorrelations). Forty-three (43) target points were developed. In orderto break the correlation between pH and HCO₃ ⁻ !, the partial pressureof CO₂ was varied. PCO₂ values were limited to a physiological range of5-50 mmHg, thus limiting the achievable values for HCO₃ ⁻ ! at a givenpH to 5-50 mmol/L. The relationship between pH and O₂ sat. was brokenthrough variation of the partial pressure of oxygen (PO₂) between 22 and480 mmHg. By using the foregoing nearly orthogonal design, correlationbetween pH and HCO₃ ⁻ !, as well as pH and O₂ sat., was minimized. Theresults are illustrated in FIG. 1 (which illustrates the relationshipbetween pH and HCO₃ ⁻ ! for the lysed blood) and FIG. 2 (whichillustrates the relationship between pH and O₂ sat. for the lysedblood).

Forty-three (43) samples of the above identified lysed blood wereprepared for analysis by spectroscopy. Each sample was a mixture ofapproximately 5 mls lysed blood, and 2 mls of a saline mixture. Theabsolute amount of lysed blood used in the mixture was adjusted in orderto obtain a hemoglobin concentration of 10 g/dL in each of the finalsamples. The saline mixture consisted 0.9% (weight) NaCl and either HClor NaOH, the choice and amount of which depended on the desired bloodgas values. The amount of acid or base required was estimated using theSigaard-Anderson equation for base excess.

Base Excess=(1-0.014 Hb!) {( HCO₃ ⁻ !)-24)+(9.5-1.63 Hb!)(pH-7.4)} whereHb! is the hemoglobin concentration in g/dL and HCO₃ ⁻ ! is thebicarbonate ion concentration in mmol/L. Base excess estimates theamount of acid or base needed to titrate blood to a normal acid basestatus (pH=7.40, pCO₂ =40 mmHg, Hb=15 g/dL, temperature=37° C.).

Each of the 43 solutions was prepared by weight. Once mixed, eachsolution was equilibrated for 10 minutes with a humidified mixture ofN₂, O₂ and CO₂ (Air Liquide, local distributor) using an IL Instrumentstonometer, model 237. Humidification is needed to prevent desiccation ofthe solutions. The samples were maintained at 37° C. (approximatelynormal human body temperature) while in the tonometer. N₂, O₂ and CO₂mixtures for the tonometer were set using a Cameron Instruments (PortAransas, Tex.) mass flow controller, model GF-3. Component ranges were6.8-7.8 for pH, 5-50 mmol/L for HCO₃ ⁻ ! and 85-100% for O₂ sat.

Once prepared, each sample was, with apparatus 11 (see FIG. 3), infusedinto a flow system that allowed for spectroscopic sampling. The flowsystem consists of a syringe pump 13 (Harvard Apparatus, South Natick,Mass.) and a 1 mm spectroscopic cell 15 connected by stainless steeltubing 17. Blood gas parameters pH, pCO₂, and pO₂ were measured(offline) prior to and after spectroscopic measurement using a Corningmodel 288 automated blood gas analyzer. No significant difference wasnoted between the two offline ABG measurements. Flow was maintained at 2ml per minute to insure that flow cell 15 remained full for 2 minutes,and to allow a 2 minute spectral acquisition which provided a low noisesignal. Prior to each new sample, the flow system was cleaned usingwater, bleach and a detergent solution. The entire flow path, excludingthe syringe pump 13, cell 15, tubing 17, and a waste container (notshown), is enclosed in a temperature controlled box 19 maintained at 37°C.

The spectral data used for the analysis described below was obtainedfrom Nicolet Magna 750 FTNIR spectrometer 21 configured for externalsampling. A 2 mm liquid nitrogen cooled InSb detector 23 was used tocollect light after transmission through the 1 mm path length of flowcell 15. Spectrometer 21 and detector 23 are optically interconnected byconventional optics, including lenses 25, 27, 29, 31 and 33. The lightsource 35 is a stabilized tungsten halogen source.

For each of the 43 samples, 356 spectral interferograms were collectedvia FTIR data acquisition accessories 37 and 39 and transformed tosingle beam scans using a DEC station 3000 (work station 41). Theresulting single beam scans were coadded resulting in one single beamspectrum for each of the 43 samples. Absorbance data were calculatedusing the resulting single beam spectra and the spectrum of cell 15(when empty) as the background. The coadditions and absorbancecalculations were performed using personal computer 43. After cuttingoff wavelengths from 2400-2500 nm (because of high noise), the resultingspectral data covered the range 1300-2400 nm with an approximateresolution of 3 nm (16 cm-1). Solutions were run in a random manner withrespect to the component (i.e., pH, HCO₃ ⁻ !, and O₂ sat) concentrationsin order to avoid correlation with instrument drift. For each of the 43spectra, 356 interferograms were coadded. Of the 43 spectra collected,39 were used for further analysis. The remaining four spectra were notincluded in the analysis because they had associated component valuessignificantly different from expected target values, putting themoutside the desired component ranges (i.e., outliers).

For comparison with the data outlined above from lysed blood and todemonstrate the relationship between pH in tissue and histidine, a 200mmol/L solution of histidine (Sigma Chemical) was prepared in saline(0.9 wt% NaCl) and titrated. Forty-four solutions were prepared spanningthe pH range 5-8. The pH of each solution was measured using a glasselectrode (Orion Research) and near infrared spectra immediatelycollected. Solutions were placed in a 10mm quartz cell and placed in thesample compartment of a Bomem MB-155 FTNIR spectrometer equipped with aInAs thermoelectrically cooled detector. Samples were run in a randomorder to prevent spurious correlation to run order. Spectra of the emptycell were collected and used for processing the solution spectra toabsorbance.

Finally, and again for comparison with the data obtained above fromlysed blood and to demonstrate that plasma proteins are not asignificant indicator species for pH, fresh frozen plasma was obtainedfrom United Blood Services (Albuquerque, N.Mex.). Once thawed, theplasma solution was centrifuged to remove any precipitated proteins. Theprotocol described above for the lysed blood samples was used forpreparation of the plasma samples (except for sonication). Forty-threesamples were prepared using the same procedure as used for the lysedblood. Spectra were collected using the apparatus illustrated in FIG. 3.Interferograms collected were processed to absorbance data prior tomathematical modeling.

With the spectroscopic data obtained above for the lysed blood,histidine and plasma, mathematical modeling (via personal computer 43)was employed to identify the spectral regions where pH can be predictedat clinically useful accuracies and that histidine provides thenecessary variation for pH modeling. PLS modeling was performed usingsoftware developed at Sandia National Laboratories. PLS is aquantitative multivariate calibration technique which models thecovariance of the spectral intensities with the reference values of theanalyte. See, D. M. Haaland and E. V. Thomas, "Partial Least-SquaresMethods for Spectral Analysis. 1. Relation to Other QuantitativeCalibration Methods and the Extraction of Qualitative Information:,Anal. Chem., 60, 1193-1202, 1988. The use of PLS and other multivariatecalibration techniques in noninvasive analysis of arterial blood gasesis discussed in the Thomas et al. patent, supra.

FIG. 4 illustrates the spectra obtained from the 39 lysed bloodsolutions identified above at varying pH, HCO₃ ⁻ ! and O₂ sat. Theprimary absorbances seen are these from water combination bands (i.e.,1420 and 1914 nm). The 39 spectra used for analysis from the lysed bloodexperiment are shown in FIG. 4 and 5 as, respectively, absorbance dataand mean centered absorbance data. Spectral variation due to pH changesare within the line widths of the individual spectra when the data aredisplayed as absorbance in FIG. 4. Primary absorbances at 1420 nm and1914 nm in FIG. 4 are due to combination bands of water, v₁ +v₃ and V₂+v₃ respectively, where v, refers to the symmetric stretching, v₂ thebending and V₃ the antisymmetric stretching mode. See, K. Buijs and G.R. Choppin, "Near-infrared Studies of the Structure of Water I. PureWater", J. Chem. Phys., 39, 2035-2041, 1963. No discernible bands fromspecies other than water are seen within the mean centered data.

PLS modeling, as explained in D. M. Haaland and E. V. Thomas, "PartialLeast Squares Methods for Spectral Analyses. 1. Relation to OtherQuantitative Calibration Methods and the Extraction of QualitativeInformation", Anal. Chem., 60, 1193-1202, 1988, aided not only indeveloping quantitative models for pH, but also as a tool for discerningthe location of information related to pH. Several spectral subregions(chosen based on the known location of carbon-hydrogen andnitrogen-hydrogen bands) were used as input into the PLS software. The1820-2040 nm region was removed because of high water absorbance,resulting in low signal to noise. Summary results are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        PLS Results for Lysed Blood Spectra                                                     Number of Cross Validated                                           Spectral Region,                                                                        Optimum   Standard Error of Prediction,                             nm        PLS Factors                                                                             pH units         R.sup.2                                  ______________________________________                                        1300-2380 5         0.175            0.506                                    2040-2380 5         0.185            0.445                                    1500-1820 13        0.037            0.978                                    ______________________________________                                    

With the instrumentation of FIG. 3, only one of the three spectralregions identified in Table 1 provided clinically useful calibrationmodels (i.e., using the data between the main water absorbances (i.e.,1420 and 1914 nm) and PLS modeling provided the best calibration modelwith a cross-validated standard error of prediction of 0.037 pH units).Clinically useful being defined as SEP values less than 0.05 pH units.The results from the 1500-1820 nm region are illustrated in FIG. 6, inwhich the reference pH values (obtained, as indicated above, with theCorning 288 automated blood gas analyzer) are plotted against thepredicted pH values using PLS modeling. Information located between thewater bands is primarily associated with carbon-hydrogen andnitrogen-hydrogen moieties, suggesting that variation in protein bandsis providing the source of the information for the pH model.

With the set up illustrated in FIG. 3, only the model for the 1500-1820nm region is clinically useful. The present data for the 2040-2380 nmregion does not provide a PLS model as strong as the model created for1500-1820 nm, primarily due to the increased noise from theinstrumentation used in acquiring the data. However, since the 2040-2380nm region has similar overtones and combination bands, with differentinstrumentation clinically useful models can be developed for allregions identified in Table 1.

In addition to PLS modeling as discussed above, other multivariatemethods (e.g. PCR, PLS2, CLS, Q-matrix, ridge regression,cross-correlation, Kalman filtering, MLR, neural networks, and continuummethods are expected to provide comparable SEP values. For example, PCRmodeling of the same data from the 1500-1820 nm region is illustrated inFIG. 7. The reference pH values plotted are the same as those used inFIG. 6.

In order to determine the source of the pH information obtained from thelysed blood, PCA (principle component analysis) analysis was performedusing the lysed blood spectral data. PCA analysis decomposes thespectral data into linear independent vectors, orthogonal to oneanother. This decomposition is done such that the maximum variance inthe data is placed in the first loading vector. Each loading vector hasassociated with it a set of scores, one score for each sample. The scorevalue for a sample represents the amount of the corresponding loadingvector contained in the sample spectrum. The scores from each loadingvector were compared to the reference pH values. The squared correlationcoefficient (R²) was calculated between each set of scores and pH andplotted. The results are illustrated in FIG. 7 where, for the 1500-1820nm region, the squared correlation coefficient (R²) calculated betweenreference pH values is plotted against eigenvector scores. Theeigenvector that provided scores giving the largest correlationcoefficient was examined and compared to those from other data sets. Asseen in FIG. 7, the scores of eigenvector 5 provide the highestcorrelation coefficient to pH. Eigenvector 5 from the lysed blood datais shown in FIG. 8. Upon examination of FIG. 8, prominent variation isnoted in the eigenvector at 1600 nm. The variation at 1600 nm isconsistent with variation C--H or N--H overtones, possibly associatedwith variation in amino acid or protein structure.

With reference to FIG. 9, a likely source of the variation seen at 1600nm in the lysed blood data is the amino acid histidine. Within theimportant physiological pH range of 6.8-7.8, the most importantionizable amino acid is histidine. See, J. E. Sherwin and B. B.Bruegger, Clinical Chemistry, Theory, Analysis and Correlation, 388-402,C. V. Mosby, St. Louis, 1984. The pK (i.e., the pH at which theconcentration of the acidic form of the molecule is equal to theconcentration of the basic form of the molecule) of the free amino acidis 5.97, however when bound, the pK can vary. The ionization occurringbetween pH 6.8 and 7.8 is: ##STR1##

In whole blood, the primary buffer (i.e., that hemoglobin which is ableto absorb more hydroxyl and hydrogen ions than any other chemicalspecies in blood and not change the pH) is the hemoglobin molecule. Thebuffering capacity of hemoglobin is determined by its histidine. Of the36 histidyl within the hemoglobin structure, 22 of these are titratable.See, C. Ho and I. M. Russu, "How Much Do We Know about the Bohr Effectof hemoglobin?", Biochemistry, 26, 6299-6305, 1987. The spectra from thetitrated histidine solutions were examined by PLS as well as PCA and theresults were compared to those from lysed blood.

Also shown in FIG. 9 is the eigenvector best correlated to pH ascalculated from the histidine spectra obtained from the set of 200mmol/L solutions described above. There are features common to botheigenvectors. In particular, similar variations at 1600 nm are seen.Chemically, the transition occurring in the histidine molecule involvesprimarily the N--H bond as the histidine is titrated. However, it isknown that the neighboring C--H bonds of the heterocycle are affected aswell. See C. T. Craescu, C. Schaeffer, J. Mispelter, J. Garin and J.Rosa, Journal of Biological Chemistry, 261, 7894, 1986. Thus thevariation seen in the eigenvector could be due to the C--2 or C--4protons or perhaps a combination of those and the N--H overtones.

In order to determine if the pH variation seen in the lysed bloodspectra is derived from primarily histidine from hemoglobin or fromhistidine in other proteins, plasma protein solutions were examined. PLSmodeling was performed using the resulting spectra. Models wereestablished, but the quantitative precision of the PLS model for pH inplasma is poor. Standard errors of predictions for the spectral region1300-2500 nm were no greater than R² =0.590 and SEP=0.231. A tableshowing the results is below:

                  TABLE 2                                                         ______________________________________                                                  Number of Cross Validated                                           Spectral Region,                                                                        Optimum   Standard Error of Prediction,                             nm        PLS Factors                                                                             pH units         R.sup.2                                  ______________________________________                                        1300-2380 6         0.259            0.483                                    2040-2380 1         0.333            0.146                                    1500-1820 6         0.231            0.590                                    ______________________________________                                    

Compared with the data set forth in Table 1 this finding suggests thatplasma proteins do not contribute significantly to the pH modeldeveloped for the lysed blood data. Further evidence of this wasobtained from the PCA analysis of the spectral data, analogous to thatperformed on the lysed blood and histidine spectral data as discussedabove. Though not illustrated no single set of scores provided a squaredcorrelation coefficient greater than 0.40. The eigenvector for lysedblood (number 5) best correlated to pH (R² ≅0.8) is shown overlaid withthose from the histidine (vector number 2) and plasma solutions (vectornumber 4) in FIG. 9.

A general shape seen in the plasma eigenvector that mimics the histidineand lysed blood eigenvectors can be seen, suggesting that similarchemistry may be present in the plasma solutions, but the variations arenot strong enough to form a quantitative model. Titration data availablefor albumin, the largest constituent in plasma, confirm that over the5-8 pH range very little buffering capacity is present. See C. Tanford,S. A. Swanson and W. S. Shore, "Hydrogen Ion Equilibria of Bovin SerumAlbumin:, J. Amer. Chem. Soc., 77, 6414-6420, 1955.

Given the fact that absorbance bands in the near infrared are overtonesand combinations of fundamental absorbance bands, near infrared bands,arising from the same fundamental mode can occur at various locationsthroughout the 500-2500 nm region. Thus, the histidine bands present at1600 nm are expected to appear at other locations. Indeed, previous workhas shown bands from histidine appearing between 2040 nm and 2380 nm,namely, 2060-2115 nm, 2160 nm, 2225-2235 nm and 2360 nm. See, Mary K.Phelan, Investigations of Quilibrium Conditions Using Near InfraredSpectroscopy, (1990) (unpublished Ph.D disseration, University ofWashington). As discussed above, the 2040-2380 nm region containshistidine information, however the instrumental system used in acquiringthe data did not allow full recovery of the signal.

To demonstrate that pH can be measured within the wavelength region1000-1300 nm, data was collected from whole blood static samples withinstrumentation (not shown) including a Perkin Elmer-2000 FT-NIRequipped with a 5 mm Ge liquid nitrogen cooled detector, and a 10 mmpathlength cuvette. Separate models were prepared for the regions1000-1300 and 1500-1820 primarily due to differences in optimalpathlength for each region. The 1000-1300 nm region contains histidinebands which are much weaker than the 1500-1820 nm bands, thus allowing alonger pathlength to be used when acquiring data. In addition, onedetector system is not sensitive to both regions simultaneously, and infact, for the present data, two different detectors were used to acquiredata from each of the regions. PLS modeling using data from the1000-1300 nm data provided results comparable to those obtained with1500-1820 nm, in particular, the SEP using the 1000-1300 nm data was0.038 with an R² =0.978. The results are illustrated in FIG. 10, inwhich reference values (obtained by conventional wet chemistry) areplotted against the predicted pH values using PLS modeling. PCA analysiswas also performed using the 1000-1300 nm data collected and compared todata collected from histidine solutions (prepared in the mannerpreviously indicated) from the same frequency region. The loadingvectors whose scores provided the largest R² to measured pH are shown inFIG. 11. Comparable variations are seen in the loading vectors, againsuggesting that the spectral signature which correlated to pH is fromhistidine.

With reference to FIG. 12, the preferred noninvasive pH monitor 111 ofthe present invention includes a spectrometer 113, an electronics andcomputer processing module 115, and a visual display module 117.Spectrometer 113 includes a broad band tungsten halogen light source119, a focusing mirror 121, a fiber optic interface 123, a second fiberoptic interface 125, a grating 127, an Insb array detector 129, and anelectronic buss 131. Module 115 includes a computing unit which, inturn, contains a microprocessor, memory, the data preprocessingalgorithms, the multivariate calibration model, the multivariatealgorithm, and outlier detection algorithms. Visual display module 117includes a pH display 141. As illustrated, display 141 includes adetermination and the estimated uncertainty of such determination. Alsoincluded in module 117 are a pH trend display 151 and a light 161 toindicate if the pH which has just been determined is an outlier.

To transmit light from spectrometer 113 to the fingertip 171 of thepatient being monitored, monitor 111 includes a source fiber opticbundle 173, which terminates at finger/fiber device 175. Receiving fiberoptic 177 returns the light from finger/fiber holder 175 to fiber opticinterface 125. Finger/fiber holder 175 allows transmission throughfinger 171 and allows for adequate holding of the finger.

In operation, source 119 emits selected frequencies from approximately1000-2500 nm. This light is focused on the end of fiber optic 173 heldin interface 123 via focusing mirror 121 and then transmitted via sourcefiber 173 to illuminate the tissue, bone, nail and blood in fingertip171. The portion of the light which is transmitted through fingertip 171is then returned to spectrometer 113 by fiber bundle 177. The returninglight is then separated into various wavelengths and detected by thearray detector 129, capable of detecting at least some of thewavelengths of light between 1300 to 2400 nm. For the frequency range1000-1300 nm it is preferred to use an InGaAs detector, otherwise theapparatus is the same.

The reflected light intensities at the various frequencies are thenanalyzed by the microprocessor of module 115, employing a multivariatealgorithm (preferably PLS) utilizing several wavelengths from variousregions of the entire spectral range of the transmitted light. Forinvasive monitoring, a system based on the apparatus of FIG. 3 or suchas described in reference to FIG. 50 in Thomas, et al. (insofar as itrelates to pH monitoring), could be utilized.

Whereas the drawings and accompanying description have shown anddescribed the preferred embodiment of the present invention, it shouldbe apparent to those skilled in the art that various changes may be madein the form of the invention without affecting the scope thereof.

What we claim is:
 1. A method of determining pH in blood containingtissue noninvasively, in vivo, and within and, physiological rangesobserved in said blood containing tissue utilizing tissue spectra whichspectra contains histidine information, said method comprising stepsof:a. generating light at three or more different wavelengths, saidwavelengths being in the range of 1000 nm to 2500 nm; b. irradiatingsaid blood containing tissue with said wavelengths so that there isdifferential attenuation of at least some intensities of saidwavelengths, said wavelength dependent differential attenuation being afunction of said blood containing tissue, including histidine in saidblood containing tissue; c. measuring at least a portion of saidintensities of said wavelengths emerging from said blood containingtissue to obtain a set of at least three spectral intensities v.wavelengths; and d. estimating said value of said pH from said measuredintensities by utilizing wavelength dependent differential attenuationderived from said histidine, said value being within said physiologicalranges.
 2. The method as set forth in claim 1, utilizing wavelengthdependent differential attentuation derived from histidine, whichexhibits a sensitivity due to changes in pH.
 3. The method of claim 1,wherein said wavelengths used for said estimation of pH are those knownto exhibit a differential attentuation due to hydrogen ions reactingwith histidine.
 4. The method as set forth in claim 3, wherein saidwavelengths are in the range of 1000-1300 nm.
 5. The method as set forthin claim 4, wherein said wavelengths include one or more of 1042, 1111and 1163 nm.
 6. The method as set forth in claim 3, wherein saidwavelengths are in the range of 1300-2500 nm.
 7. The method as set forthin claim 6, wherein said wavelengths are in the range of 1500-1820 nm.8. The method as set forth in claim 7, wherein said wavelengths include1600 nm.
 9. The method as set forth in claim 6, wherein said wavelengthsare in the range of 2040-2500 nm.
 10. The method as set forth in claim9, wherein said wavelengths include one or more of 2060-2115, 2160,2225-2235 and 2360 nm.
 11. A quantitative analysis instrument fornoninvasive spectroscopic measurement of pH in human tissue, saidinstrument comprising:a. a source of at least three differentwavelengths of light, said wavelengths being in the range of 1000-2500nm, at least some of said wavelengths having a wavelength dependentdifferential attenuation due to histidine; b. optics for directing saidwavelengths into said blood containing tissue; c. at least one detectorfor measuring the intensity of at least a portion of said wavelengthsemerging from said blood containing tissue that are differentiallyattenuated by histidine; d. electronics for processing said measuredintensities derived from said wavelengths that are differentiallyattenuated by histidine to estimate pH values in said tissue; and e.means for indicating said estimated values of blood pH.
 12. The analysisinstrument of claim 11, wherein the said electronics utilizes amultivariate algorithm using at least two variables and a calibrationmodel.
 13. The analysis instrument of claim 12, wherein said calibrationmodel is developed by an algorithm that is capable of using morespectral variables per sample than the number of calibration samplesused to generate said model, and wherein each of said variablesrepresents at least one spectral intensity.
 14. The analysis instrumentof claim 11, wherein said wavelengths are in the range of 1000-1300nm.15. The analysis instrument of claim 14, wherein said wavelengthsinclude one or more of 1042, 1111 and 1163 nm.
 16. The analysisinstrument of claim 11, wherein said wavelengths are in the range of1300-2500nm.
 17. The analysis instrument of claim 16, wherein saidwavelengths are in the range of 1500-1820nm.
 18. The analysis instrumentof claim 17, wherein said wavelengths include 1600 nm.
 19. The analysisinstrument of claim 16, wherein said wavelengths are in the range of2040-2500nm.
 20. The analysis instrument of claim 19, wherein saidwavelengths include one or more of 2060-2115, 2160, 2225-2235 and 2360nm.
 21. A method of determining pH in blood, within and physiologicalranges observed in blood, utilizing spectra which spectra containshistidine information, said method comprising steps of:a. generatinglight at three or more different wavelengths, said wavelengths being inthe range of 1000 nm to 2500 nm; b. irradiating said blood with saidwavelengths so that there is differential attenuation of at least someintensities of said wavelength, said wavelength dependent differentialattenuation being a function of said blood including histidine in saidblood; c. measuring at least a portion of said intensities of saidwavelengths emerging from said blood to obtain a set of at least threespectral intensities v. wavelengths; and d. estimating said value ofsaid pH from said measured intensities by utilizing wavelength dependentdifferential attenuation derived from said histidine, said value beingwithin said physiological ranges.
 22. The method as set forth in claim21, utilizing wavelength dependent differential attentuation derivedfrom histidine, which exhibits a sensitivity due to changes in pH. 23.The method of claim 21, wherein said wavelengths used for saidestimation of pH are those known to exhibit a differential attentuationdue to hydrogen ions reacting with histidine.
 24. The method as setforth in claim 23, wherein said wavelengths are in the range of1000-1300 nm.
 25. The method as set forth in claim 24, wherein saidwavelengths include one or more of 1042, 1111 and 1163 nm.
 26. Themethod as set forth in claim 23, wherein said wavelengths are in therange of 1300-2500 nm.
 27. The method as set forth in claim 26, whereinsaid wavelengths are in the range of 1500-1820 nm.
 28. The method as setforth in claim 27, wherein said wavelengths include 1600 nm.
 29. Themethod as set forth in claim 23, wherein said wavelengths are in therange of 2040-2500 nm.
 30. The method as set forth in claim 29, whereinsaid wavelengths include one or more of 2060-2115, 2160, 2225-2235 and2360 nm.
 31. A quantitative analysis instrument for spectroscopicmeasurement of pH in blood, said instrument comprising:a. a source of atleast three different wavelengths of light, said wavelengths being inthe range of 1000-2500 nm, at least some of said wavelengths having awavelength dependent differential attenuation due to histidine; b.optics for directing said wavelengths of light into said blood; c. atleast one detector for measuring the intensity of at least a portion ofsaid wavelengths of said light emerging from said blood that aredifferentially attenuate by histidine; d. electronics for processingsaid measured intensities derived from said wavelengths that aredifferentially attenuate by said histidine to estimate pH values in saidtissue; and e. means for indicating said estimated values of blood pH.32. The analysis instrument of claim 31, wherein the said electronicsutilizes a multivariate algorithm using at least two variables and acalibration model.
 33. The analysis instrument of claim 32, wherein saidcalibration model is developed by an algorithm that is capable of usingmore spectral variables per sample than the number of calibrationsamples used to generate said model, and wherein each of said variablesrepresents at least one spectral intensity.
 34. The analysis instrumentof claim 31, wherein said wavelengths are in the range of 1000-1300nm.35. The analysis instrument of claim 34, wherein said wavelengthsinclude one ore more of 1042, 1111 and 1163 nm.
 36. The analysisinstrument of claim 31, wherein said wavelengths are in the range of1300-2500nm.
 37. The analysis instrument of claim 36, wherein saidwavelengths are in the range of 1500-1820nm.
 38. The analysis instrumentof claim 37, wherein said wavelengths include 1600 nm.
 39. The analysisinstrument of claim 36, wherein said wavelengths are in the range of2040-2500nm.
 40. The analysis instrument of claim 39, wherein saidwavelengths include one or more of 2060-2115, 2160, 2225-2235 and 2360nm.